The fusion of membranes is a common event in biological systems and nature has developed elegant mechanisms for that. For example, the infection of a cell by a virus is one event in which fusion of membranes plays a key role.
It is therefore not surprising that the ability of viruses to promote fusion with cellular or endosomal membranes led to the development of recombinant viral gene delivery systems. The most prominent systems rely on retroviral-, adenoviral-, adeno-associated viral- or herpes simplex viral vectors, which are employed in more than 70% of clinical gene therapy trials worldwide. Although virus based gene delivery systems are very efficient, they show immune-related side effects after the injection. This major drawback limits the safety of these systems and consequently their applicability in humans (e.g., Thomas et al., Nature Reviews, Genetics, 4, 346-358, 2003).
An alternative is the use of non-viral vectors to deliver genetic material into cells. Nonviral vector systems include, for example, cationic polymers and liposomes. Liposomes are artificial single, oligo or multilamellar vesicles having an aqueous core and being formed from amphipathic molecules. The cargo may be trapped in the core of the liposome or disposed in the membrane layer or at the membrane surface. Today, liposomal vectors are the most important group of the nonviral delivery systems. More specifically, cationic liposomes or lipids have been used widely in animal trials and/or clinical studies. Although cationic systems provide high loading efficiencies, they lack colloidal stability, in particular after contact with body fluids. Ionic interactions with proteins and/or other biopolymers lead to in situ aggregate formation with the extracellular matrix or with cell surfaces. Cationic lipids have often been found to be toxic as shown by Filion, et al. in BBA, 1329(2), 345-356, 1997; Dass in J. Pharm. Pharmacol, 54(5), 593-601, 2002; Hirko, et al. in Curr. Med. Chem., 10(14), 1185-1193, 2003.
Amphoteric liposomes represent a recently described class of liposomes having an anionic or neutral charge at pH 7.5 and a cationic charge at pH 4. WO 02/066490, WO 02/066012 and WO-A-03/070735, all to Panzner, et al. and incorporated herein by reference, give a detailed description of amphoteric liposomes and suitable lipids therefor. Further disclosures are made in WO-A-03/070220 and WO-A-02/066489, also to Panzner, et al. and incorporated herein by reference, which describe further pH sensitive lipids for the manufacture of such amphoteric liposomes.
Amphoteric liposomes can encapsulate nucleic acid molecules with high efficiency equal to cationic liposomes. Advantageously, amphoteric liposomes are much better tolerated upon administration in vivo and show a favourable biodistribution over cationic liposomes.
Compared to viral gene delivery vectors the non-viral systems are much safer; they are tolerated at high doses and do not elicit an immune response, therefore these systems can be administered repetitively. Still, viral systems are superior in terms of transfection efficacy. Attempts have been made to incorporate viral surface glycoproteins into liposomes (Miller N, Vile R., FASEB J, 9, 190-199, 1995) but these hybrid systems again have the drawback of immune-related side effects.
There is therefore an ongoing need for improvements of non-viral systems.
It is known in the prior art that the pH sensitive hydrophilic moieties can be used for engrafting ligands or active ingredients to a liposomes. WO-A-86/04232 of Kung et al., for example, discloses liposome compositions containing coupling groups in their outer bilayer region to bind surface bound molecules such as proteins. The coupling reagents disclosed by Kung et al. include a phosphatidylethanolamine lipid moiety, a carbon containing spacer arm of up to 20 atoms and a terminal carboxyl group. The terminal carboxyl group is employed as a coupling group, and the hydrophobic moiety provides spatial separation between the lipid bilayer and said coupling group.
A related approach is disclosed in WO-A-97/19675 of Wheeler which relates to cationic lipids of a Rosenthal inhibitor (RI) core structure having an alkyl linking group with up to 10 carbon atoms, e.g., a carboxyalkyl group. RI structures with additional carboxyalkyl groups are disclosed as intermediates for further functionalisation, e.g., with ligands for cellular uptake, therapeutic molecules or groups that can increase the polar charge density of the cationic lipids.
U.S. Pat. No. 6,294,191 describes lipids comprising long hydrophobic moieties lacking a hydrophilic group ( ). These structures do not react to changes in pH.
WO-A-00/11007 of Tournier et al. discloses phosphatidic acid esters for stabilising liposome vesicles in suspension in water, buffers and biological liquids against rupture and coalescence with time. Such esters are also said to increase encapsulating capacity and stabilise the vesicle membrane against leakage of the entrapped substances toward the carrier liquid.
EP-A-0099068 to Eibl discloses glycerol derivatives.
Prior art is silent to the use and optimization of structural elements (II) or lipids (I) to enhance cellular uptake and cytosolic delivery of liposomes and sequestered active ingredient. Prior art has not taught, that the combination of hydrophobic moieties with the pH sensitive hydrophilic moieties provides criticality to such function.